Esophageal squamous cell carcinoma marker circRNA and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an esophageal squamous carcinoma marker circRNA and application thereof, wherein the marker is hsa_circ_0001701, and the sequence of hsa_circ_0001701 is shown as SEQ ID NO. 1. The application further discloses application of the marker in preparation of an esophageal squamous carcinoma diagnosis and / or prognosis marker product. The application provides a new view for searching for a biomarker and a drug treatment target for clinical diagnosis and prognosis evaluation of esophageal squamous carcinoma, lays a foundation for related drug research, and has important significance for research and treatment of esophageal squamous carcinoma, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an esophageal squamous carcinoma marker circRNA and application thereof. BACKGROUND

[0002] The statements in this section merely provide background information related to the present application and do not necessarily constitute prior art.

[0003] Esophageal squamous carcinoma is a common malignant tumor of the digestive tract. For early and middle stage esophageal squamous carcinoma patients, surgical treatment is the preferred mode. For early esophageal squamous carcinoma, endoscopic submucosal dissection is the first-line treatment for patients whose cancer tissue is limited to the esophageal mucosa or submucosa. For advanced esophageal squamous carcinoma patients, radical surgery or neoadjuvant combined radical surgery treatment is widely accepted. Advanced esophageal squamous carcinoma patients lose the opportunity for surgery, although radiotherapy and immunotherapy and other programs prolong the survival period of advanced esophageal squamous carcinoma patients, but the 5-year survival rate is only 26%.

[0004] Due to the special circular structure of circRNA without 5' cap and 3' ploy(A) tail, circRNA has higher stability and longer half-life than linear RNA. At the same time, circRNA has tissue specificity, different contents in different tissues, and the advantage of easy detection, which can detect circRNA from body fluids such as blood, urine and saliva. circRNA has the advantage of becoming a diagnostic marker and a therapeutic target. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide an esophageal squamous carcinoma marker circRNA.

[0006] Another purpose of the present application is to provide the application of the esophageal squamous carcinoma marker circRNA.

[0007] The present application is realized by the following technical solutions:

[0008] The first aspect of the present application provides an esophageal squamous carcinoma marker circRNA, the marker is: hsa_circ_0001701. The sequence of hsa_circ_0001701 is shown in SEQ ID NO. 1.

[0009] The second aspect of the present application provides the application of hsa_circ_0001701 in the preparation of esophageal squamous carcinoma diagnosis and / or prognosis marker product. By detecting the up-regulation of hsa_circ_0001701 expression in tissues or blood, it is beneficial to evaluate the relationship between the expression level of hsa_circ_0001701 marker and esophageal squamous carcinoma.

[0010] The above-mentioned applications include but are not limited to any one of the following modes:

[0011] (1) By detecting the expression content of hsa_circ_0001701 in the patient detection sample, whether the patient is an esophageal squamous cell carcinoma patient is determined;

[0012] (2) By detecting the expression content of hsa_circ_0001701 in the esophageal squamous cell carcinoma patient detection sample, the prognosis of the patient is evaluated;

[0013] (3) The detection reagent of hsa_circ_0001701 is used for preparing an esophageal squamous cell carcinoma diagnosis and / or prognosis detection reagent.

[0014] In the application of the first aspect, the detection sample of the diagnostic marker is a clinical biological sample of a patient, including but not limited to one or more of serum (plasma), whole blood, secretion, cotton swab, pus, body fluid, tissue, organ, paraffin section, etc.

[0015] In a third aspect of the present application, a reagent combination for esophageal squamous cell carcinoma diagnosis and / or prognosis detection is provided, which is used for detecting the expression content of hsa_circ_0001701 in a detection sample.

[0016] Preferably, a diagnostic kit suitable for PCR method for detecting hsa_circ_0001701 is provided,

[0017] Further, the primer is a hsa_circ_0001701 primer to be detected; in a specific example, the circRNA primer and the internal reference primer sequence are as follows:

[0018]

[0019] Beneficial effects

[0020] The circRNA of the present application can not only be used for rapid and effective early detection of esophageal squamous cell carcinoma, but also provides a therapeutic target and important basis for gene therapy, drug therapy and other clinical applications.

[0021] The present application provides a new perspective for finding biomarkers for clinical diagnosis and prognosis evaluation of esophageal squamous cell carcinoma and drug treatment targets, lays a foundation for related drug research and development, and has important significance for the research and treatment of esophageal squamous cell carcinoma, thus having good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 For qRT-PCR verification of the expression amount of has_circ_0001701 in esophageal squamous cell carcinoma and normal epithelial tissue adjacent to the cancer;

[0023] Figure 2 For has_circ_0001701 expression and clinical relevance;

[0024] Figure 3 For qRT-PCR verification of has_circ_0001701 expression in esophageal squamous cell carcinoma cell lines;

[0025] Figure 4 For has_circ_0001701 overexpression and knockdown efficiency verification in TE-1 cell line;

[0026] Figure 5 For EdU experiment to verify that has_circ_0001701 promotes the proliferation of esophageal squamous cell carcinoma cells;

[0027] Figure 6 For CCK8 experiment to verify that has_circ_0001701 promotes the proliferation of esophageal squamous cell carcinoma cells;

[0028] Figure 7 For colony formation experiment to verify that has_circ_0001701 promotes the proliferation of esophageal squamous cell carcinoma cells;

[0029] Figure 8 For has_circ_0001701 structure diagram. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be further explained and described below through specific examples. The experimental supplies used in the present application are all commercially available.

[0031] Example 1

[0032] (1) circ_0001701 differential expression in esophageal cancer and clinical relevance analysis. Clinical samples (Qilu Hospital of Shandong University, all breast cancer and cancer adjacent tissues were obtained during surgery. All participants have obtained written informed consent. This study was approved by the Ethics Committee of Qilu Hospital of Shandong University)

[0033] Extract 45 pairs of esophageal squamous cell carcinoma and cancer adjacent normal epithelial tissue RNA, and verify has_circ_1701 (SEQ ID NO. 1) found in sequencing to be significantly overexpressed in cancer tissue by qRT-PCR.

[0034] The operation process is as follows:

[0035] (1) RNA extraction

[0036] The tissue RNA extraction kit used in this experiment is produced by Takara Company, and RNA extraction is performed according to the instructions. The operation process is as follows:

[0037] ① Take soybean-sized tissue and place it in a mortar. Grind the tissue under liquid nitrogen conditions to prevent the specimen from melting. Collect the tissue powder and transfer it to an enzyme-free EP tube.

[0038] ② Add 50x DDT solution to the lysis buffer, then add 350 μL of lysis buffer to each enzyme-free EP tube, mix repeatedly by pipetting to ensure sufficient lysis, and let it stand at room temperature for 2 minutes.

[0039] ③ Place the gDNA adsorption column on the collection tube, then transfer the lysate to the gDNA adsorption column, centrifuge at 4°C, 12000 rpm for 1 min, and collect the filtrate.

[0040] ④ Add 350 μL of 70% ethanol to the filtrate and mix thoroughly.

[0041] ⑤ Transfer the mixed solution to the RNA adsorption column, centrifuge at 12000 rpm at 4°C for 1 min, and discard the filtrate.

[0042] ⑥ Add 500 μL of Buffer RWA to the RNA adsorption column, centrifuge at 12,000 rpm at 4°C for 30 seconds, and discard the filtrate.

[0043] ⑦ Add 600 μL of Buffer RWB to the RNA adsorption column, centrifuge at 12,000 rpm at 4°C for 30 seconds, and discard the filtrate.

[0044] ⑧Prepare DNase I reaction solution: take 41 μL RNase-free dH2O, 5 μL 10x DNase I Buffer and 4 μL Recombinant DNase I, mix thoroughly, and then add dropwise to the center of the RNA adsorption column. Let it stand at room temperature for 15 minutes.

[0045] ⑨ Add 350 μL of Buffer RWB to the center of the RNA adsorption column, centrifuge at 12,000 rpm at 4°C for 30 seconds, and discard the filtrate.

[0046] ⑩Repeat step ⑦.

[0047] The RNA adsorption column was placed back into the collection tube and centrifuged at 12,000 rpm at 4°C for 2 min.

[0048] Transfer the RNA adsorption column to a new 1.5 mL enzyme-free EP tube, add 50 μL of enzyme-free water to the center of the RNA adsorption column, and let it stand at room temperature for 5 minutes.

[0049] Centrifuge at 12,000 rpm for 2 min at 4°C to obtain RNA, which can be frozen or used for the next step.

[0050] (2) circRNA reverse transcription PCR

[0051] The circRNA reverse transcription kit used in this experiment was produced by Shanghai Yisen Biotechnology Co., Ltd. After measuring the RNA concentration, the RNA reverse transcription was carried out according to the instructions, and the operation process was as follows:

[0052] ① Remove gDNA. Configure the following reaction solution in a 200 μL enzyme-free EP tube, mix thoroughly, and incubate at 42°C for 2 min.

[0053]

[0054] ② Prepare 20 μL reverse transcription system.

[0055]

[0056] ③ After the reverse transcription system is prepared, mix thoroughly, and reverse transcribe according to the following program.

[0057]

[0058]

[0059] (3) qRT-PCR

[0060] The reverse transcription kit used in this experiment was produced by Shanghai Yisen Biotechnology Co., Ltd.

[0061] ① Prepare 20 μL reaction system on ice, and the specific components are as follows:

[0062]

[0063] ② Add the prepared reaction system to eight rows, and set three repeated holes for each sample. According to the instructions, set up three-step amplification.

[0064]

[0065] ③ β-catin as the internal reference gene, according to 2 -ΔCT The relative expression amount of the gene was calculated according to the formula.

[0066] The circRNA primer and internal reference primer sequence are as follows:

[0067]

[0068]

[0069] Figure 1In the middle, blue represents the relative expression of circRNA in normal esophageal epithelial tissue, and red represents the relative expression of circRNA in esophageal squamous cell carcinoma samples. β-actin as internal reference. Cancer: esophageal squamous cell carcinoma samples; Normal: normal esophageal epithelial tissue. The results show that the expression of has_circ_0001701 in esophageal squamous cell carcinoma tissue is significantly higher than that in paracancerous tissue (P=0.0011). P<0.05 is considered statistically significant. Figure 1

[0070] Example 2

[0071] The median of the relative expression of has_circ_0001701 in esophageal squamous cell carcinoma samples was used as the dividing line, and the first 23 patients with relative expression were divided into has_circ_0001701 high expression group, and the rest of the patients were has_circ_0001701 low expression group. The clinical data of the patients were obtained from the hospital information management system, including age, gender and pathological results.

[0072] Figure 2 The expression of has_circ_0001701 affects the prognosis of patients with esophageal squamous cell carcinoma. Low: has_circ_0001701 relative low expression group; High: has_circ_0001701 relative high expression group. Survival analysis showed that the survival time of patients in the has_circ_0001701 high expression group was significantly lower than that in the has_circ_0001701 low expression group.

[0073] Example 3

[0074] In order to further verify the expression of has_circ_0001701 in esophageal normal epithelial cells and esophageal squamous cell lines, the RNA of HET-1A, Eca-109, TE-1, KYSE-410, KYSE-150 and KYSE-30 cells was extracted, and the relative expression of has_circ_0001701 was detected by qRT-PCR after reverse transcription.

[0075] Figure 3 The relative expression of has_circ_0001701 in esophageal normal epithelial cells and esophageal squamous cell lines, the results showed that has_circ_0001701 was significantly up-regulated in the detected esophageal squamous cell lines. The TE-1 cell line with the most obvious up-regulation of expression was selected for subsequent experiments.

[0076] Example 4

[0077] ​To further verify the effect of has_circ_0001701 expression on the biological function of esophageal squamous cell carcinoma cells, has_circ_0001701 overexpression plasmid, blank control plasmid (Vector) and siRNA, siRNA-NC were transfected into TE-1 and KYSE-150 cell lines, respectively.

[0078] Figure 4 A. has_circ_0001701 overexpression efficiency verification; B. has_circ_0001701 knockdown efficiency verification. has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA#1: small interference sequence 1 group; has_circ_0001701 siRNA#2: small interference sequence 2 group; siRNA-NC: small interference control group. It was found that after transfection of has_circ_0001701 overexpression plasmid or recombinant lentivirus, qRT-PCR showed that the relative expression of has_circ_0001701 was significantly increased; and after transfection of shRNA recombinant lentivirus, qRT-PCR showed that the relative expression of has_circ_0001701 was significantly reduced, while the expression of FNDC3B was not significantly changed.

[0079] In TE-1 cell line, the effect of has_circ_0001701 overexpression plasmid and siRNA on the proliferation of esophageal squamous cell carcinoma cells was verified by EdU, CCK8 and colony formation experiments.

[0080] Example 5

[0081] EdU experiment: EdU is a thymidine analogue that can be taken up by cells. During cell proliferation, it can replace normal thymine and integrate into new DNA to form EdU-DNA complexes, which can be visualized under a fluorescence microscope after binding with a fluorescent dye, thereby realizing the detection of cell proliferation ability. In this study, the EdU kit produced by Shanghai Biyun Tian Biotechnology Co., Ltd. was used for experiments according to the instructions, and the operation process was as follows:

[0082] (1) Reagent preparation

[0083]

[0084]

[0085] (2) Experimental process

[0086] ① Count the cells to be tested, and dilute them to 2×10 5Cells were seeded in 96-well plates at a density of cells / well, and then incubated overnight in a cell incubator after shaking.

[0087] ②EdU labeling. Dilute the EdU solution to 10 μM at a ratio of 1:1000, according to the volume of medium per well 100 μL, prepare the appropriate amount of EdU working solution, and preheat the prepared working solution in a 37°C water bath. Discard the old medium, and add 100 μL of the prepared EdU working solution to each well. Incubate in the incubator for 2 h.

[0088] ③After the EdU labeling is completed, discard the medium, rinse the cells with an appropriate amount of PBS buffer once, and add 50 μL of 4% paraformaldehyde to fix the cells for 15 min at room temperature.

[0089] ④Aspirate the fixing solution, add an appropriate amount of washing solution to each well to rinse the cells for 3 min, and repeat 3 times.

[0090] ⑤Aspirate the washing solution, add 100 μL of permeabilization solution to each well, and incubate at room temperature for 15 min. Remove the permeabilization solution, and then add an appropriate amount of washing solution to rinse the cells for 3 min, and repeat 2 times.

[0091] ⑥Add 50 μL of Click reaction solution to each well, shake well, and then incubate at room temperature for 30 min in the dark.

[0092] ⑦Aspirate the Click reaction solution, add an appropriate amount of washing solution to each well to rinse the cells for 3 min, and repeat 3 times.

[0093] ⑧Aspirate the washing solution, add 100 μL of Hoechst working solution to each well, shake well, and then incubate at room temperature for 10 min in the dark.

[0094] ⑨Aspirate the Hoechst working solution, add an appropriate amount of washing solution to each well to rinse the cells for 3 min, and repeat 3 times. Then take photos under a fluorescence microscope. Red fluorescence represents EdU-positive cells, and blue fluorescence represents DAPI-labeled cell nuclei. Randomly select 3 fields of view per well for observation and photography, and the EdU-positive rate = (red fluorescent cells / blue fluorescent cells) x 100%.

[0095] Figure 5 In the middle, the EdU experiment represents the graph and the result statistics graph, and the scale = 100 μm. has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA #1: small interfering sequence 1 group; has_circ_0001701 siRNA #2: small interfering sequence 2 group; siRNA-NC: small interfering control group. EdU experiment shows that the has_circ_0001701 overexpression group has a higher proportion of proliferating cells, while knockdown reduces the proportion of proliferating cells.

[0096] Example 6

[0097] CCK8 experiment: CCK-8 experiment is a common method for detecting cell viability and proliferation capacity. Its effective component is tetrazolium salt which can be metabolized by living cells into yellow formazan salt. The metabolite is positively correlated with cell viability and quantity. The viability and quantity of the measured cells can be evaluated by detecting the optical density of the metabolite. The experimental procedure is as follows:

[0098] (1) Count the cells to be tested, inoculate 2000 cells per well into a 96-well plate, 5 repeated wells per well, 4 repeated plates per plate, and incubate overnight in a cell incubator.

[0099] (2) CCK-8 solution preparation.

[0100]

[0101] (3) Discard the culture medium, add 100 μL of 10% CCK-8 staining solution to each well, and incubate in the incubator for 2 h in the dark.

[0102] (4) After incubation, use a multifunctional enzyme marker to detect the optical density value (OD value) at 450 nm.

[0103] (5) Measure a repeated plate every 24 h, and draw a curve.

[0104] Figure 6 CCK8 experiment results curve of has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA #1: small interference sequence 1 group; has_circ_0001701 siRNA #2: small interference sequence 2 group; siRNA-NC: small interference control group. The CCK8 experiment curve shows that overexpression of has_circ_0001701 improves cell growth rate and activity, and the knockdown group is opposite.

[0105] Example 7

[0106] Colony formation experiment:

[0107] (1) Count the cells to be tested, inoculate 1.5 x 10^3 cells per well into a 6-well plate, set 3 repeated wells per group, shake well, and incubate in a cell incubator for 10-12 days. During the period, observe the cell morphology and change the liquid as needed.

[0108] (2) After obvious cell colonies are formed, the culture medium is aspirated, the PBS buffer is used for rinsing, 1 mL of tissue fixing solution is added into each well, and the cells are fixed at room temperature for 20 min.

[0109] (3) The tissue fixing solution is aspirated, the PBS buffer is used for rinsing, 1 mL of 0.1% crystal violet solution is added, and the cells are dyed at room temperature for 30 min.

[0110] (4) The crystal violet is recovered, the PBS buffer is used for rinsing for 3 times, the double-distilled water is used for rinsing for 2 times, the liquid is discarded, and the cells are naturally dried.

[0111] (5) The cells are placed under a shadowless lamp for photographing, and the number of formed colonies is counted.

[0112] Figure 7 The colony formation experiment represents a graph and a result statistical graph, has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA #1: small interference sequence 1 group; has_circ_0001701 siRNA #2: small interference sequence 2 group; siRNA-NC: small interference control group. The colony formation experiment shows that more cell clone groups are formed by overexpressing has_circ_0001701, and the number of clone groups is reduced after knocking down has_circ_0001701.

[0113] The above merely describes preferred embodiments of the present application but is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of primers for detecting hsa_circ_0001701 in preparing a product for the diagnosis and / or prognosis of esophageal squamous cell carcinoma, wherein the primer sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3.

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